Extraction and Stability of Monascus Pigments from Fermentation Broth of Mona...

奥鹏网院作业

Extraction and Stability of Monascus Pigments from Fermentation Broth of Monascus purpureus YY1-3 Using Ethanol/Ammonium Sulfate Aqueous Two-Phase SystemExtraction and Stability of Monascus Pigments from Fermentation Broth of Monascus purpureus YY1-3 Using Ethanol/Ammonium Sulfate Aqueous Two-Phase System

KONG Weibao1, YANG Shuling1, YANG Yang2, CHEN Dong1, GUO Baomin1, ZHANG Aimei1, NIU Shiquan1

(1. College of Life Science, Northwest Normal University, Lanzhou 730070, China;2. Jinhui Wine Co. Ltd., Longnan 742500, China)

Abstract: Monascus pigments (MPs) were extracted from the fermentation broth of Monascus purpureus YY1-3 in a simple and efficient way using an ethanol/ammonium sulfate aqueous two-phase system (ATPS). Thin layer chromatography (TLC)was used to analyze the composition of the MPs and their stability was also tested. The results showed that when the ATPS was composed of 16% (m/m) ethanol and 28% (m/m) ammonium sulfate, the maximum partition coefficient and recovery rate of MPs of 171.91 and 98.13% were obtained respectively. When the volume proportion of the top phase extraction solution-ethanol was 1:0.5, ammonium sulfate could be effectively removed from it. TLC showed that the extract was composed of six components, mainly Monascus red and yellow pigments. The MPs were stable in darkness, pH 4–8 and 25–65 ℃, as well as in the presence of common metal ions and food additives.

Keywords: Monascus pigments; Monascus purpureus; extraction; aqueous two-phase system; stability

Color and flavor are the signals that are immediately perceived by the optical and chemical senses of humans and these attributes determine whether or not a certain food is appealing. Recent increasing concern on the use of edible coloring agents has led to ban a variety of synthetic coloring agents, which have potential carcinogenicity and teratogenicity. Thus, it is necessary to develop natural, green and low cost processes for the production of pigments for replacing the synthetic ones due to high cost of currently used technology of pigment production on an industrial scale. This circumstance has inevitably increased demands for highly safe, naturally occurring edible coloring agents, one of which is Monascus pigments (MPs)[1-2]. MPs are a group of fungal secondary metabolites called azaphilones, which have similar molecular structures and chemical properties. It has been known that MPs as natural food colorants have been widely utilized in food industries in the world, especially in China,Japan, and southeastern Asian countries[3-4]. Moreover, MPs have a range of biological activities, such as anti-mutagenic,anticancer properties and antimicrobial activities[5-9].

由于我国现有的森林资源有限，能源面临着短缺的局面。因此，在进行森林资源的合理开发利用过程中，在保证森林资源经济效益的基础上，要注重森林资源开发利用的生态效益，只有做到生态效益与经济效益的协调发展，才能够实现森林资源的合理利用。此外，在森林资源的开发利用过程，要基于长远发展的角度考虑开发问题，制定科学合理的开发方案，体现出森林资源开发的合理性。基于我国可持续发展战略目标下，必须结合目前森林资源的实际现状，适当的调整森林资源开发利用战略目标，不但要保证其更好的适应我国经济发展的需要，而且还要充分发挥其生态功能，调节环境气候，为人类的生存发展提供重要的环境基础，使其为我国社会的发展发挥其作用。

Solid-state fermentation (SSF) and liquid-state fermentation (LSF) are two major processes for MPs production. MPs produced by LSF possesses have many advantages, including shorter cultivation time, lower production costs, higher product quality and easier management [10-13]. Therefore, the MPs were produced by LSF in this paper. MPs could be divided into water-soluble and water-insoluble constituents according to their solubility in water. Most of which are water-insoluble. The total MPs are usually extracted by ethanol at various concentrations, the extraction of water-insoluble MPs constituents is achieved with organic solvents such as n-hexane, benzene, methanol,ethanol, and the water-soluble MPs are taken by distilled water[2]. However, the yields of MPs extracted by all the separation methods mentioned above were relatively low.

Aqueous two-phase extraction is a kind of separation technology which can obtain high recovery and purity by using simple equipment and operating under mild conditions,and thus, has the advantages of high resolution, quick phase separation, possibility of continuous operation, simple scaleup and low energy consumption[14-18]. The traditional aqueous two-phase system (ATPS) is polymer/polymer or polymer/salt system, which is suitable for extraction of biological macromolecules with polarity, such as protein and enzyme.However, the low partition coefficient and the high cost of the polymer have made it unsuitable for industrial application.Compared with the traditional two-aqueous system, short chain alcohol/salt system has clearer phase separation, lower cost, and the extraction phase does not contain high viscosity and difficult to handle polymers[19-20]. In this study, therefore,we selected an ethanol-ammonium sulfate system to extract MPs from crude broth fermented by Monascus purpureus(M. purpureus) with high coefficient and recovery yield.Ethanol and ammonium sulfate are two water-soluble substances which are not compatible with each other. The competition of inorganic molecules for water molecules leads to the enrichment and phase separation of organic molecules.MPs are mostly soluble in ethanol solution, so MPs are distributed in the top phase of the ATPS. However, some ammonium sulfate was dissolved in the top phase. In order to purify and concentrate MPs in the later stage, ammonium sulfate in the top phase needed to be removed. The components and effects of light, pH, temperature, common metal ions and food additives on the stability of the MPs were also investigated. aiming to provide experimental data for its further development and utilization in food industry.

1 Materials and Methods1.1 Materials and reagents

The strain M. purpureus YY1-3 was isolated and obtained by microbiology laboratory of College of Life Sciences, Northwest Normal University (China). Ethanol was purchased from Shuangshuang Chemical Reagent Co. Ltd.(Yantai, Shandong, China). Ammonium sulfate was obtained from Guangfu Technology Development in China. Hydrogen peroxide, citric acid, sodium nitrite and other chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd.(Shanghai, China).

The fermentation medium (per liter) was comprised of defined amounts of glucose 60 g, peptone 20 g, MgSO4·7H2O 1.0 g, KH2PO4 1.0 g, NaNO3 2.0 g, MnSO4 0.1 g, and ZnSO4·7H2O 0.1 g, at natural pH.

1.2 Instruments and equipments

AC2-4S1 Biosafety cabinet (Singapore Art High); FCD-3000 Constant temperature blast drying oven (Shanghai langgan Experimental Equipment Co. Ltd.); TG20-WS Refrigerated centrifuge (Hunan Xiangli Scientific Instrument Co. Ltd.); UV-2800 Ultraviolet visible spectrophotometer(Unocal Instruments Co. Ltd.); GI54TW Automatic pressure steam sterilizer (Zhiwei Instruments Co. Ltd.); HNY-2102C Intelligent constant temperature cultivation oscillator (Tianjin Ono Instrument Co. Ltd.); scientz-12ND Freeze dryer(Ningbo Xinzhi Biotechnology Co. Ltd.); HCJ-6D Digital display constant temperature water bath (Jintan Guowang experimental instrument factory); meter S20 Micro pH(Mettler Toledo, Switzerland).

所有图像由2名具有丰富影像诊断工作经验的副主任职称以上的医师共同阅片，意见不一致时经协商达成统一意见。评价项目包括：病变部位、形态、密度、生长方式、与周围结构关系、有无骨质改变及区域淋巴结肿大。8例病变术前均诊断恶性肿瘤性病变。

1.3 Methods

1.3.1 Fermentation parameters and crude sample preparation

领导小组下设办公室，办公地点设在市水务局。办公室主任由市水务局主要负责同志担任，副主任由各有关部门负责同志担任。办公室负责贯彻领导小组的决定和部署，明确工作任务，草拟管理标准、制度和考核办法，监督各项任务的落实，组织实施考核等工作，定期公布经领导小组审定的考核结果。

The crude sample used in this study was obtained from batch fermentation of M. purpureus YY1-3 under aerobic fermentation at 30 ℃ in a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium for 7 days with shaking speed of 200 r/min. After 7 days of fermentation,the fermentation liquid was filtered to remove insoluble impurities and mycelium, and the crude extract of MPs was obtained and used for further tests.

藤尾因为苦于先进的现代女性观，但是被利己主义和本位主义所束缚。在这反复的纠葛中，藤尾也不知道如何选择。这是藤尾选择死亡的直接原因。藤尾经过一系列心理的崩溃，失去希望，悄然死去。其他人持续着悲喜交加的生活，全文也落下帷幕。

1.3.2 Preparation of ATPS and extraction

A finite quantity of ammonium sulfate was dissolved in water, and then certain volumes of ethanol and crude extract were added into the ammonium sulfate solution, and mixed well to form two phases. After that, more water was added to make the total weight of the system equal to 10 g.The mixture was vortexed thoroughly and centrifugalized at 5 000 r/min for 5 min to obtain clear phase separation. Then,the top and bottom phases were collected and analyzed with colorimetric method.

The concentration of MPs was determined using the following function (equation (1)).

由于风力发电机组多安装在高山、荒野、海岛等风口处，受无规律的变向变载荷的风力作用以及强阵风的冲击，加之所处的自然环境交通不便，而且机组安装在塔顶的狭小空间内，一旦出现故障，修复非常困难，故对其可靠性等提出了比一般机械高得多的要求。大量的实践证明，整个机组的薄弱环节常常就是齿轮箱。因此，风力发电机组齿轮箱在投入使用前，都会进行严格的试验，以确保其高可靠性。而测试齿轮箱的变频电动机，必须经受齿轮箱的各种复杂工况，这也就对电动机提出了较高的要求。

   

Where A505 nm and C are the absorbance value and concentration of MPs/ (mg/mL), respectively.

The partition coefficient of the MPs in ATPS was calculated using the equation (2)[21].

“支架”由Wood et al.(1976)提出，指学生在完成特定任务时教师给予的支持。在社会文化理论角度下，“支架”理论与最近发展区理论密切相关。

   

Where Ct and Cb are the concentrations of MPs in the top and bottom phass respectively.

The phase ratio is defined as the ratio of volume of top phase to that of the bottom phase (equation (3)).

对着镜子自拍出马甲线、A4腰的时候，健身达人的背景通常是健身房，哑铃、杠铃和各种健身器械摆在一起，配图里少不了健身餐。朋友圈中的“轻食套餐”往往有着相似的配置：两片黑木耳、三只虾、几根青菜叶，加上切碎的鸡胸肉，整整齐齐地码在一起。

Vessel Traffic Flow Prediction Based on FCM-ANFIS Model

   

Where Vt is the volume of the top; Vb is the volume of the bottom.

The extract recovery rate was defined as the ratio to the concentration of MPs in the top phase (mg/mL) to the MPs concentration in crude extract (mg/mL) (equation (4)).

   

Where Ct and Vt are the concentration and volume of MPs in the top phase; Cb and Vb are the concentration and volume of MPs in the bottom phase.

1.3.3 Identification of MPs composition by thin layer chromatography (TLC)

The developing agent (trichloroethane-methanol=1:1,V/V) was prepared and transferred to the developing tank to seal the chromatography cylinder for 60 min, so that the developing agent was evenly distributed in the whole space of the chromatography cylinder. M. purpureus YY1-3 fermentation liquid extracted by aqueous two-phase was spotted on TLC Silica GF254 plates (Merck, 5 cm × 20 cm)with a micropipette (Eppendof) with strip spot method. The strip was then air dried for 5 min before development. The plates were developed in 25 mL of trichloromethane-methanol(1:1, V/V) for approximately 60 min. The plates were dried for 5 min and viewed under a UV lamp. TLC detection was undertaken in triplicate for samples.

1.3.4 Partial characterization and stability test

Effect of pH: The effect of pH variation on the stability of MPs was studied on pH values: 3.0, 4.0, 5.0, 6.0, 7.0,8.0 and 9.0. The procedures were followed as described:1 mL of MPs extracted by ATPS was mixed with 10 mL of prepared ethanol in each of the three valcon test tubes.The tubes were placed in a dark room and kept at room temperature for 5 days.

Effect of light treatment: 1 mL of MPs extracted by ATPS was diluted with 10 mL of prepared ethanol and the MPs before light treatment (darkness, natural light, lamplight,UV, indoors and outdoors) were determined at 505 nm wavelength. For light stability, 1 mL of MPs extract were placed in each of the three valcon test tubes and placed under different light treatments for 0, 1, 2, 3, 4 and 5 hours.

地点分布分别是手术室32例次(65.31%)，病室13例次(26.53%)，供应室、伤员分类平台各1例次(分别占2.04%)，其他舱室2例次(4.08%)。

Effect of heat treatment: 1 mL of MPs extracted by ATPS was diluted with prepared ethanol and the MPs before heating were determined at 505 nm wavelength. For heat stability, 1 mL of MPs extract was placed in each of the three valcon test tubes and heated in a thermostatically controlled water bath at 25, 45,65, 85, and 100 ℃ for 0, 1, 2, 3, 4, 5 and 6 hours.

银行内部机构设置雍杂，部门职能重复，不能对人员和资源进行有效配置，一方面造成员工薪酬的增加，另一方面造成资源浪费成本增加。整合银行部门，解放人力，减少业务及管理费支出，提高服务效率，增加营业收入。建立合理的薪酬激励机制，基于绩效考评体系建立薪酬制度，根据贡献程度，进行薪酬的相应增加。还需了解竞争对手薪酬变化情况，以确保银行薪酬水平保持动态竞争力。良好的员工薪酬体系可以提供“人尽其才”的软性环境，在银行内部形成“能者上庸者下”的良好氛围。既能减少业务及管理费支出，也能提高银行的盈利能力。

Effect of metal ions and food additives: 1 mL of MPs extracted by ATPS was diluted with prepared ethanol and the MPs before treatment were determined at 505 nm wavelength.For metal ions and food additives stability, 5 mmol/L metal ions (KCl, NaCl, CaCl2, FeCl3, CuSO4 and ZnCl2) and 200 of food additives (glucose, sucrose, sodium benzoate, citric acid,glacial acetic acid and sodium nitrite, hydrogen peroxide,ascorbic acid, sorbic acid, dehydroacetic acid and sodium propionate) were added to 1 mL of MPs extract for 0, 1, 2, 3,4, 5, 6 and 7 days.

1.4 Analysis and calculation methods

Absorbance was measured at 505 nm wavelength.The concentration of MPs was determined by colorimetric method[22-23]. Data were reported as ± s of triplicate determinations. Origin 9 Statistics and SPSS Statistics 22 software were used to analyze the data.

2 Results and Analysis2.1 Effect of ethanol and ammonium sulfate concentrations on the extraction rate of MPs

The salt and solvent characteristics affected the formation of phase. To select the appropriate ratio of ethanol to ammonium sulfate in ATPS, we added the crude extracts to the different phase forming salts with different ethanol solutions. The phase formation range of the ethanolammonium sulfate system is wide, with the ethanol mass fraction of 7%-78% and the ammonium sulfate mass fraction of 0.15%-38%. Therefore, considering the separation effect and cost comprehensively, the concentration range of ethanol was determined to be 15% to 40%, and the concentration range of ammonium sulfate was 20% to 30% (Table 1).

The crude extracts were added to ATPS with ammonium sulfate and ethanol at several concentrations. The effect of ethanol mass fractions on the recovery rate of MPs was tested, while the ammonium sulfate was kept constant. As can be seen in Table 1, the mixtures of ammonium sulfate(20%)-ethanol (15%) and ammonium sulfate (25%)-ethanol(15%) were added to the crude extract of fermentation,but no stratification occurred in the solution. However,the extraction system of ammonium sulfate (30%)-ethanol(15%) was stratified, and the recovery rate and partition coefficient were both high. This could be due to the reason that the concentration of ammonium sulfate was too low to allow the pigment molecules in the water to be all separated out. The results showed that the increasing concentrations of ammonium sulfate could cause an increase in partition coefficients and recoveries for MPs, and a slight decrease in volume ratio. Therefore, we can optimize the extraction system by adjusting the concentration of ammonium sulfate.Moreover, the formation of the two phases was affected by the hydration ability of the solvent, which was related to the solvent structure. In general, a higher polarity solvent can bind more water molecules and was thus superior for MPs extraction.

In addition, it can be seen from Table 1 that the recovery rates of the stratified system were all higher, but their volume ratios and partition coefficients were different, among which recovery rates and partition coefficients of ammonium sulfate(25%)-ethanol (20%), ammonium sulfate (30%)-ethanol(15%), ammonium sulfate (30%)-ethanol (20%) and ammonium sulfate (30%)-ethanol (30%) were higher.However, among the above four different extraction systems,the smallest volume ratio was observed in ammonium sulfate(30%)-ethanol (15%) as compared to those of the other investigated extraction systems. Hence, the purity of MPs recovered from this extraction system was the highest.

Table 1 Effects of ethanol concentration on recovery rate of MPs,partition coefficient and volume ratio of ethanol

     

Notes: —. No layering. The same as below.

Recovery rate/%12015－－－2 20 20 19.00 ± 0.23 26.28 ± 0.41 96.85 ± 1.64 3 20 25 1.67 ± 0.04 46.83 ± 0.74 98.90 ± 2.11 4 20 30 1.38 ± 0.02 69.64 ± 0.57 98.96 ± 1.01 5 20 35 1.50 ± 0.07 55.00 ± 0.98 98.81 ± 1.79 6 20 40 2.33 ± 0.11 62.5 ± 1.31 99.32 ± 3.17 72515－－－8 25 20 0.54 ± 0.02 117.45 ± 2.61 98.45 ± 1.43 9 25 25 0.73 ± 0.14 102.54 ± 1.01 98.68 ± 0.95 10 25 30 1.11 ± 0.11 103.45 ± 3.5399.14 ± 2.15 11 25 35 1.64 ± 0.26 40.88 ± 2.44 98.53 ± 1.67 12 25 40 2.70 ± 0.09 47.17 ± 1.79 99.22 ± 3.05 13 30 15 0.20 ± 0.01 300.87 ± 2.45 98.38 ± 1.46 14 30 20 0.46 ± 0.08 116.57 ± 1.69 98.18 ± 2.68 15 30 25 0.73 ± 0.06 100.43 ± 1.7898.66 ± 1.52 16 30 30 1.11 ± .012 156.74 ± 2.7199.43 ± 3.11 17 30 35 1.71 ± 0.05 15.88 ± 0.49 96.44 ± 2.93 18 30 40 2.51 ± 0.01 96.95 ± 1.88 99.59 ± 0.68 NumberAmmonium sulfate/% Ethanol/%Volume ratio Partition coefficient

Effect of the added amounts of ammonium sulfate on the recovery rate of MPs was tested according to the results mentioned above. As shown in Table 2, the recovery rate of MPs was increased with the increase of ammonium sulfate concentration from 15% to 30%. In addition, when the ammonium sulfate concentration was low, 25% ethanol has poor phase-separating effect, and the recovery rate of MPs was low. With the increase of ammonium sulfate, the recovery rate of 25% ethanol was increased greatly, while those of the other groups were increased slowly. In general,a slower increase in the recovery rate of MPs occurred by increasing concentration of ammonium sulfate than by increasing the concentration of ethanol. It is due to the reason that the change in recoveries was an integrated result of volume ratio and partition coefficient. Paraphrasing, with the ammonium sulfate concentration increasing, the water molecules get into the bottom phase, and consequently attract the MPs to the bottom phase.

It can be seen from Table 2, the recovery rates and partition coefficients of ethanol (25%)-ammonium sulfate(24%), ethanol (25%)-ammonium sulfate (27%), ethanol(25%)-ammonium sulfate (30%), ethanol (30%)-ammonium sulfate (24%) and ethanol (30%)-ammonium sulfate (30%)were all higher among the three different ethanol solutions.However, the highest MPs recovery rate (99.51%) was observed in 35% ethanol solution compared to other investigated ethanol solutions. Furthermore, it can be observed that all of the partition coefficients of the MPs are great, implying that the target compounds are preferentially partitioned to the top phase.

Table 2 Effects of ammonium concentration on recovery rate of MPs,partition coefficient and volume ratio of ethanol

     

NumberEthanol/%Ammonium Recovery rate/%12515－－－2 25 18 0.82 ± 0.02 10.52 ± 0.1189.60 ± 1.04 3 25 21 0.64 ± 0.01 65.74 ± 0.3597.67 ± 0.91 4 25 24 0.67 ± 0.12122.83 ± 0.6198.79 ± 2.11 5 25 27 0.64 ± 0.08117.20 ± 0.4398.69 ± 1.11 6 25 30 0.67 ± 0.02102.40 ± 0.3798.56 ± 3.01 7 30 15 1.50 ± 0.11 24.38 ± 0.1697.34 ± 0.61 8 30 18 1.22 ± 0.21 98.48 ± 0.3199.18 ± 1.24 9 30 21 1.22 ± 0.33 98.21 ± 0.6699.17 ± 1.09 10 30 24 1.11 ± 0.45120.73 ± 0.9599.26 ± 2.01 11 30 27 1.26 ± 0.01 76.70 ± 1.1199.03 ± 1.99 12 30 30 1.07 ± 0.19119.19 ± 2.0199.22 ± 1.67 13 35 15 1.86 ± 0.04 60.78 ± 0.8799.12 ± 3.22 14 35 18 1.50 ± 0.34133.82 ± 0.6799.51 ± 1.68 15 35 21 1.38 ± 0.29 97.89 ± 1.0499.27 ± 1.35 16 35 24 1.63 ± 0.12 57.36 ± 0.5598.85 ± 2.01 17 35 27 1.71 ± 0.11 78.66 ± 1.0699.26 ± 1.92 18 35 30 1.71 ± 0.09 40.00 ± 0.5498.56 ± 0.78 sulfate/% Volume ratio Partition coefficient

2.2 Optimization of the ATPS composition

Based on the above results, the system of MPs extraction with ethanol-ammonium sulfate was further optimized by orthogonal test. From the results, it was evident that the recovery rate and partition coefficient of MPs under each extraction system were all high. Among them, the highest partition coefficient of MPs were obtained at ethanol (18%)-ammonium sulfate (28%) and ethanol(16%)-ammonium sulfate (32%). However, the recovery rate of ethanol (16%)-ammonium sulfate (28%) was high, and the volume ratio was the lowest and purity was the highest.Therefore, considering the cost, ethanol (16%)-ammonium sulfate (28%) and ethanol (18%)-ammonium sulfate (28%)systems were selected to conduct a scale-up experiment to study the extraction method of MPs. The results of MPs extraction in the orthogonal experiment were shown in Table 3.

根据已有的研究结果[3-5]，按多元logistic回归模型中样本容量的计算方法，利用2013年第五次国家卫生服务调查重庆农村地区的相关数据，失访率以50%计，计算样本容量为270人。实际共抽得高血压患者280人。利用Epidata录入问卷调查数据，数据整理和分析采用统计工具软件stata.11。

Table 3 Results of optimization of ammonium sulfate and ethanol concentration in ATPS using orthogonal experimental design

     

NumberAmmonium sulfate/% Ethanol/%Volume ratio Partition coefficient Recovery rate/%1 26 16 0.27 ± 0.02 99.59 ± 1.0196.37 ± 0.08 2 26 18 0.37 ± 0.04110.82 ± 0.9397.61 ± 0.56 3 26 20 0.56 ± 0.11 84.75 ± 0.2297.93 ± 1.21 4 26 22 0.52 ± 0.02 85.85 ± 0.4398.80 ± 0.46 5 28 16 0.31 ± 0.05171.91 ± 0.6198.13 ± 1.09 6 28 18 0.36 ± 0.05219.51 ± 0.7398.74 ± 0.92 7 28 20 0.48 ± 0.02 53.42 ± 0.2696.39 ± 1.11 8 28 22 0.61 ± 0.04 54.67 ± 0.7897.08 ± 0.59 9 30 16 0.37 ± 0.05 83.75 ± 0.9396.87 ± 0.22 10 30 18 0.43 ± 0.09107.29 ± 1.0997.88 ± 0.37 11 30 20 0.48 ± 0.02 62.11 ± 1.0196.78 ± 0.46 12 30 22 0.59 ± 0.04 49.69 ± 0.5996.78 ± 0.38 13 32 16 0.37 ± 0.04 200 ± 0.45 98.66 ± 0.98 14 32 18 0.42 ± 0.02135.48 ± 1.2198.37 ± 0.59 15 32 20 0.48 ± 0.07106.71 ± 0.6898.16 ± 0.45 16 32 22 0.64 ± 0.05 72.07 ± 0.9497.88 ± 1.04

The scale-up experiment results of ethanol-ammonium sulfate aqueous two-phase extraction of MPs were shown in Fig. 1. The results showed that the recovery rates and partition coefficients of MPs of ammonium sulfate (28%)-ethanol (16%) were 98.93% and 196.07, and ammonium sulfate (28%)-ethanol (18%) were 99.09% and 203.95, among which the minimum volume ratio and the highest purity were obtained at ammonium sulfate (28%)-ethanol (16%) was selected as the optimized ATPs of MPs.

     

Fig. 1 Pictures of 16 orthogonal array runs (A) and scale-up experiments (B)

2.3 Recycle of ammonium sulfate in the top phase

It is worthwhile pointing out that a large amount of ammonium sulfate was used in this separation system.Although ethanol can be easily recovered by distillation, the recycling of salt in this ATPS is also a problem that needed to be resolved. Ammonium sulfate in the top phase can be recovered by adding ethanol, as shown in Fig. 2. When the volume ratio of the top phase extraction solution-ethanol was increased from 1:0 to 1:1.5, the weight of the ammonium sulfate recovered was increased from 0.024 g to 0.194 g.However, when the volume ratio of ethanol reached 0.75, the weight of the ammonium sulfate recovered was 0.144 g. So the optimal volume ratio of the top phase extraction solutionethanol was determined to be 1:0.5.

     

Fig. 2 Effect of ethanol proportion in the top phase on the recovery rate of ammonium sulfate

The above results showed that it was feasible to separate MPs from fermentation broth using an ethanol-ammonium sulfate ATPS. The extraction process not only extracted MPs effectively, but also recycled ethanol and ammonium sulfate in the ATPS through distillation and dilution crystallization of ethanol, respectively. This process is also easy to scale-up.

2.4 Partial purification of MPs using spectroscopic identification

MPs components were isolated and purified by column chromatography, TLC, high-performance liquid chromatography, capillary electrophoresis, and high-speed counter-current chromatography[24-25]. TLC is simple to operate, fast to analyze, and does not require expensive instruments. Its results are intuitive, and it has high separation ability, especially in the detection of a large number of samples. Thus, it is widely used in the analysis of complex samples. Monascus pigment is a secondary metabolite of Monascus. It is frequently used in various literatures to separate MPs by TLC. In 1973, monascin and ankaflavin were isolated from mycelia of M. anka through TLC using 25% ether in benzene as developing agent[26]. Therefore, the selection of TLC for the separation of fermentation liquid extracted by aqueous two-phase has also become the first experiment optional. The results of TLC in MPs isolation were listed in Table 4 and Fig. 3.

但媳妇还是不放心。她把脱了一半的体恤又穿上，趿拉着鞋，到院子里看院墙是不是够高，能不能翻进人来。又检查门栓插好没有，锈蚀得厉害不厉害，能不能禁得住外人的拉扯。玻璃是不是遮挡的严实，能不能从缝隙窥视到里面的情景……媳妇这么折腾一气，回来了。躺在床上还是不踏实，身子僵硬得像块木板。在新居中的第一夜并没像他想象的那么美好，被媳妇的紧张情绪破坏了。他现在什么心情也没有，只想睡他娘的觉！

Monascus red was used as the contrast and trichloroethane-methanol (1:1) as the developing agent to separate the fermentation broth extract of M. purpureus YY1-3. The results showed that the extract contained three components of red pigment with Rf values of 0.653, 0.713,and 0.747; and three components of yellow pigment with Rf values of 0.727, 0.807 and 0.865, respectively (Table 4). Under the condition of natural light and UV, different pigment bands were observed and their contents were evaluated,as shown in Fig. 3. Compared with the control group, the fermentation broth extract contained the most basic Monascus red pigment while Monascus contained the yellow pigment.The three bands of red pigment were relatively clear, but their contents were different. The band with Rf value of 0.747 was heavier in color, indicating higher content. The separation effect of the three bands of yellow pigment was better, but the proportion of yellow pigment in the total pigment was smaller, so the bands of yellow pigment were lighter.

Table 4 TLC characteristics of MPs

     

First band 0.653 Red, thin Second band 0.713 Red, thin Third band 0.727 Yellow, thin Fourth band 0.747 The natural light: red, thick, bandwidth; UV light:clear and thick. High content Fifth band 0.807 Yellow, thin Sixth band 0.865 The natural light: yellow, thick, bandwidth; UV light:clear and thick. High content

     

Fig. 3 Separation of MPs by TLC

2.5 Stability of MPs

Consumers are concern to replace the synthetic additives by natural products. However, due to the instability of these compounds, difficulties, such as natural pigments with functional properties, may be encountered. In order to overcome the instability problem of these bioactive compounds, optimizing storage conditions has become an important tool, helping to increase shelf life and protecting the biological properties of the material.

2.5.1 Effects of lights and temperatures

     

Fig. 4 Effects of light (A and B) and temperature (C) on the stability of the extract

The MPs are sensitive to light, especially to sunlight and ultraviolet lights, and the yellow MPs constituents are more photostable than the red MPs ones[27-28]. The effects of light treatment (darkness, natural sunlight, lamplight,UV, indoor and outdoor) on MPs extract were studied by exposed the extract under different lights. Meanwhile,thermal stability of the extract was studied at 25, 45, 65,85 and 100 ℃ for holding times of 0, 1, 2, 3, 4, 5, 6 h. The data obtained were given in Fig. 4. The retention rate of the extract were decreased slightly and then tended to be stable under darkness, natural sunlight, and lamplight. However,the retention rate was decreased rapidly and then tended to be stable within 0 to 5 h after exposed to UV light. The effect of the extract on the stability under indoor, outdoor and darkness were shown in Fig. 4B. Under indoor condition, the retention rate of the extract was decreased rapidly and then tended to stabilization. Within 0-4 d, there was a downward trend, and within 4-5 d, the retention rate basically remained stable.Under outdoor condition, the retention rate of the extract was decreased first and then leveled off. Under the condition of darkness, the retention rate of the extract was decreased slightly, but remained at about 96%.

The effects of different temperatures (25, 45, 65, 85 and 100 ℃) on the stability of MPs extract were shown in Fig.4C. The retention rate of the extract was stable after heat treatment at 25, 45, 65 ℃ for 0 to 6 hours, but when heating temperature was elevated up to 85 ℃, the retention rate was decreased rapidly. Therefore, the temperature over 65 ℃ will be conducive to the preservation of MPs.

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2.5.2 Effects of pH

Usually, MPs are very stable at 30-60 ℃ and pH 6.0-8.0[12]. But some MPs are still stable even at higher temperatures and extreme pH values. Ji Hao et al.[29] reported that MPs from M. anka were still relatively stable at pH 11.0.The stability of M. purpureus YY1-3 fermentation liquid extracted by aqueous two-phase was determined at a range of pH values between 3 and 9, by measuring the retention rate of MPs at 0 to 5 d (Fig. 5). In the range of 0-5 d, the stability of the pigments was affected by different pH values. During the period from 0 to 5 d, the retention rate of the extract decreased with the extension of time. Among them, the pH that had the least impact on the stability of the extract was 5 and 6, and the pH that had the greatest impact on the stability of the extract was 3 and 9.

     

Fig. 5 Effect of pH on the stability of the extract

2.5.3 Effects of metal ions and food additives

Metal ions can also affect the MPs stability to some extent. Frequently, MPs are stable in the presence of a small quantity of Na+, Mg2+, K+, Al3+, Ca2+, Cu2+, and Zn2+, but the Fe3+ and Fe2+ showed an obvious negative effect on stability of MPs at the concentrations of 20, 40, 100 mg/L[30-31]. Metal ions stability of MPs was studied by adding different metal ions for holding times of 7 days, the retention rate of the extract was measured. As can be seen from Fig. 6A, the extract was stable under the condition of adding KCl, NaCl,CaCl2, ZnCl2 and CuSO4 at 30 ℃ for 7 days, but when FeCl3 was added, the retention rate of the extract was decreased dramatically. The above results indicated that Fe3+ had a greater negative influence on the stability of MPs.

In the same way, different food additives (glucose,sucrose, sodium benzoate, citric acid, glacial acetic acid,sodium nitrite, hydrogen peroxide, ascorbic acid, sorbic acid,dehydroacetic acid and sodium propionate) were added and stored at 30 ℃ for 7 days. The retention rate of the extract was measured daily to study the effects of different food additives on the stability of MPs (Fig. 6B, C, D). At the time of 0-1 d, the retention rate of the extract was decreased with 6 different additives, among which the rate was decreased the fastest when glacial acetic acid was added. With the extension of time, except that the retention rate of the extract of glacial acetic acid showed a trend of continuous decline, the retention rate of the extract was basically stable of the other 5 different additives. It can be seen from the above results that the least impact on the stability of MPs was found in the addition of citric acid and sodium benzoate, while a greater influence on the stability of MPs was found in the addition of glucose, sucrose and sodium nitrite, and the largest influence on the stability of the extract was found with the addition of glacial acetic acid.

   
     

Fig. 6 Effect of metal ions (A) and food additives (B, C and D) on the stability of the extract

It can be seen from Fig. 6C that ascorbic acid (VC) has little influence on the stability of MPs. At the time of 0-1 d,the retention rate of the extract added with VC was decreased slightly. During 1-7 d, the stability of the extract was basically maintained in a stable state. However, the retention rate of the extract added with hydrogen peroxide (H2O2)was decreased rapidly at 0-1 d, and the stability of the MPs was closed to 0 at 1-7 d. Fig. 6D showed the retention rate of the extract after adding preservatives. After the addition of dehydroacetic acid, sodium propionate and sorbic acid,the effects of those food additives on the retention rate of MPs was not obvious with the extension of time. The above results showed that the oxidant (H2O2) had adverse effects on the stability of MPs, suggesting that it is necessary to avoid contact with oxidants when using MPs.

3 Conclusion

This work reported a simplified strategy to separate MPs from fermentation broth using an ammonium sulfate-ethanol ATPS. The optimized system was composed of 28% (m/m)ammonium sulfate and 16% (m/m) ethanol, yielding partition coefficient 171.91, volume ratio 0.31 and recovery rate of 98.13% for MPs, respectively. Most of the target products(MPs) can be partitioned efficiently into the top phase, which can be easily purified via further processes. In addition, the ammonium sulfate in the top phase can also be recovered by the addition of ethanol. The effectiveness of this separation method coupled to its lower cost and recyclable nature may have great potential for industrial applications.

M. purpureus YY1-3 fermentation liquid extracted by ATPS was analyzed by TLC. The result indicated that the yellow and red components were produced during fermentation. Furthermore, the stability of MPs under different light, pH, temperature, metal ions and food additives on the stability was studied. The results showed that the MPs were stable under darkness, pH 4-8, 25-65 ℃,as well as in conventional metal ions and food additives solution. Therefore, the adverse effects of strong light,high temperature, extreme pH conditions, iron ions and oxidant should be avoided as far as possible in the storage of MPs. The above results will be of great significance for the extraction and preservation of MPs, which will greatly promote its application in food industry.

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乙醇-硫酸铵双水相萃取紫色红曲菌发酵液中的红曲色素及其稳定性分析

孔维宝1，杨树玲1，杨 洋2，陈 冬1，郭保民1，张爱梅1，牛世全1

（1.西北师范大学生命科学学院，甘肃 兰州 730070；2.金徽酒股份有限公司，甘肃 陇南 742500）

摘 要：建立一种采取乙醇-硫酸铵双水相体系，从紫色红曲菌YY1-3菌株发酵液中简单、高效萃取红曲色素（Monascus pigments，MPs）的方法，利用薄层色谱（thin layer chromatography，TLC）分析提取液的色素成分，并考察了该菌株产红曲色素的稳定性。结果表明：当萃取体系中添加质量分数16%的乙醇和28%的硫酸铵时，MPs的分配系数与回收率达到最大，分别为171.91和98.13%；当上相萃取液-乙醇体积比为1∶0.5时，可有效分离回收上相中的硫酸铵；TLC分析结果表明，萃取液中有6 种主要色素组分，其中以红曲红和红曲黄色素为主；色素稳定性实验结果表明，在避光、pH 4～8、25～65 ℃以及常规金属离子和食品添加剂溶液中，该菌株所产的MPs具有良好的稳定性。

关键词：红曲色素；紫色红曲菌；提取；双水相体系；稳定性

收稿日期：2019-04-08

基金项目：国家自然科学基金地区科学基金项目（31360192）；陇原青年创新创业团队项目

第一作者简介：孔维宝（1981—）（ORCID: 0000-0001-6151-5962），男，副教授，博士，研究方向为微生物技术。E-mail: kwbao@163.com

DOI:10.7506/spkx1002-6630-20190408-095

中图分类号：TS202

文献标志码：A

文章编号：1002-6630（2020）08-0091-09

引文格式：

KONG Weibao, YANG Shuling, YANG Yang, et al. Extraction and stability of Monascus pigments from fermentation broth of Monascus purpureus YY1-3 using ethanol/ammonium sulfate aqueous two-phase system[J]. 食品科学, 2020, 41(8):91-99. DOI:10.7506/spkx1002-6630-20190408-095. http://www.spkx.net.cn

KONG Weibao, YANG Shuling, YANG Yang, et al. Extraction and stability of Monascus pigments from fermentation broth of Monascus purpureus YY1-3 using ethanol/ammonium sulfate aqueous two-phase system[J]. Food Science, 2020, 41(8):91-99. DOI:10.7506/spkx1002-6630-20190408-095. http://www.spkx.net.cn